Non-molting dwarf (nm-d) as a mutant of Bombyx mori with a defect in purine synthesis
Non-molting dwarf (nm-d) as a mutant of Bombyx mori with a defect in purine synthesis
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DOI：
10.1016/j.ibmb.2021.103636
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发表时间：
2021-09-21
影响因子：
3.8
通讯作者：
Banno, Yutaka
中科院分区：
文献类型：
--
作者：
Fujii, Tsuguru;Kakino, Kohei;Banno, Yutaka
家蚕有几种已知的非蜕皮突变，包括非蜕皮侏儒（nm-d）。具有这种突变的幼虫正常孵化并开始吃叶子，但在第一次蜕皮完成之前死亡。nm-d突变的遗传分析将有助于分离鳞翅目昆虫幼虫发育必需的基因。为了鉴定nm-d基因座的致病基因，我们进行了基于RNA-seq的粗略定位。使用两组RNA-seq数据，一组来自正常幼虫的合并样品，一组来自nm-d幼虫的合并样品，将nm-d基因座缩小到500 kb区域。在位于该区域的基因中，在ATIC（5-氨基咪唑-4-甲酰胺核糖核苷酸转化酶/肌苷5 '-单磷酸环化水解酶）（BmATIC）基因的Bmx同源物中鉴定出nm-d特异性外显子缺失，所述基因催化哺乳动物中从头嘌呤生物合成途径的最后两个步骤。PCR和随后的测序分析表明，包含BmATIC基因的外显子9的区域在nm-d幼虫中缺失。使用CRISPR/Cas9系统产生的BmATIC基因的敲除等位基因（BmATIC（KO））显示，第一龄敲除幼虫死亡，同时表现出深棕色幼虫体，这是在体壁中缺乏尿酸的突变体的典型特征。致死幼虫由+/BmATIC（KO）蛾之间的杂交产生。与正常幼虫相比，nm-d幼虫一龄幼虫全身尿酸含量显著降低。这些结果表明，BmATIC基因负责nm-d表型，nm-d幼虫具有嘌呤生物合成缺陷，包括尿酸。我们还讨论了BmATIC mRNA通过母体传递给卵子的可能性。我们的研究结果表明，使用合并样品的基于RNA-seq的作图是鉴定致死突变的致病基因的实用方法。
There are several known non-molting mutations of the silkworm, Bombyx mori, including non-molting dwarf (nm-d). Larvae with this mutation hatch normally and start eating leaves, but die before the completion of the first ecdysis. Genetic analysis of the nm-d mutation would contribute to the isolation of essential genes for the larval development of lepidopteran insects. To identify the causative gene of the nm-d locus, we conducted RNA-seq based rough mapping. Using two sets of RNA-seq data, one from a pooled sample of normal larvae, and one from a pooled sample of nm-d larvae, the nm-d locus was narrowed to a 500 kb region. Among the genes located in this region, a nm-d-specific exon loss was identified in the Bombyx homolog of the ATIC (5-aminoimidazole-4-carboxamide ribonucleotide transformylase/Inosine 5'-monophosphate cyclohydrolase) (BmATIC) gene, which catalyzes the final two steps of the de novo purine biosynthetic pathway in mammals. PCR and subsequent sequencing analysis revealed that a region containing exon 9 of the BmATIC gene is deleted in the nm-d larvae. A knockout allele of the BmATIC gene (BmATIC(KO)), that was generated using the CRISPR/Cas9 system, revealed that first instar knockout larvae died while exhibiting the dark brown larval body that is a typical feature of mutants that lack uric acid in the integument. Lethal larvae resulted from crosses between +/BmATIC(KO) moths. The uric acid content in the whole-body of the first instar was drastically reduced in the nm-d larvae compared to normal larvae. These results indicated that the BmATIC gene is responsible for the nm-d phenotype, and that nm-d larvae have a defect in purine biosynthesis, including uric acid. We also discuss the possibility that the BmATIC mRNA is maternally transmitted to eggs. Our results indicated that RNA-seq based mapping using pooled samples is a practical method for the identification of the causative genes of lethal mutations.